| Botryosphaeria dothidea is a major important pathogen and widely distributed and serious damaged.to forest and fruit trees,which reported more than 20 countries in world,in china,it is the most serious disease and causes the huge losses.Understanding the molecular pathogenisis mechanism of pathogen is great significance for effectively prevention and control of B.dothidea.At present.B.dothidea mainly focus on the morphological classification,system development,genetic diversity,as well as the chemical control,but there are lack of related research for molecular pathogenisis mechanism.However the construction of mutant library and knockout the genes involved in pathogenicity are effective method for the research of pathogenic genes.Isolation of the genes involved in pathogenicity is crucial for fungal molecular biology,which may be approached by genetic mutagenesis analysis and to reveal the molecular pathogenic mechanism of B.dothidea as early as possible and provide theoretical basis and methods for disease prevention and control.In this study,using the developed ATMT mediated transformation of B.dothidea to construct mutant library and 1053 transformants were obtained.In addition,It has been evidence that T-DNA was inserted into the B.dothidea genome by several subcultures and PCR amplification.systematically analysis of the phenotypes of 526 mutant stranins and amplification the flanking sequences of the T-DNA insertio sites of some growth-related mutants were carried out.The experimental results were as follows:1.The wild type strain B.dothidea SDAU11-126 was used as control,eight mutants strains was obtained by observing the colony color,measuring the size of colony and pathogenic force measurement.The strain of LW-27 was aerial hyphae and closely but not luxuriantly,the colony was light yellow,the growth rate and pathogenic were minimal and growth rate and pathogenic have significant difference,the growth rate and pathogenicity were decreased 28.64% and 54.90%.The strain of LW-22 was aerial hyphae closely but not luxuriantly,the colony was light yellow,the growth rate and pathogenic hadn’t exist significant difference compared with wild type.The strain of LW-7 was aerial hyphae and loose and luxuriantly,colony was white in the early days and invisible green in late,thegrowth rate and pathogenic had significant difference and growth rate and pathogenicity increased for 27.60% and 13.76%.The strain of LW-31 was aerial hyphae and loose and luxuriantly,the colony was milk white,the growth rate and pathogenicity were maximum and had significant difference compared with wild type,the growth rate and pathogenicity increased 29.35% and 15.65%.The strain of LW-26 was aerial hyphae and closely but not luxuriantly,the colony was white,the growth rate was significant differences,the growth rate decreased 24.43%,but its pathogenicity did not exist significant difference.The strain of LW-64 was aerial hyphae and loose but not luxuriantly,the colony was rusty,the edge of colony appeared water soluble,the growth rate and pathogeniclty existed significant difference.The strain of LW-100 was aerial hyphae and loose but not luxuriantly,whichcolony was rusty,colony appeared annular,the growth rate and pathogenicity hadn’t exist significant difference.The strain of LW-111 was aerial hyphae and loose and luxuriantly,which colony was milk white and appeared concentric annular,the growth rate was significant differences and the growth rate increased 27.83%,but its pathogenicity hadn’t exist significant difference compared with wild type.2.Flanking sequences of 4 mutant strains were amplificating by using hi TAIL-PCR method,.The LW-27 was 994 bp and annotated as Glycerol Kinase;the flanking sequences of LW-100 was 1097 bp and annotated as GDP-Mannose Pyrophosphorylase;the flanking sequences of LW-64 was 1105 bp and annotated as GDP-Mannose Pyrophosphorylase;the flanking sequences of LW-31 was 1154 bp and annotated as hypothetical protein. |
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